Ezine

Published: Jan 13, 2014

Author: Steve Down

Channels: Sample Preparation

Protein leakage from transgenic crops

Transgenic crops are becoming commonplace despite continued protests from campaigning groups who are unsure about their safety. Although commercial products must be approved by government organisations before their dissemination, many groups believe that there are risks concerning food safety, environmental contamination and cross-pollination of crops.

One of the early genetically modified crops was maize, Zea mays, in which genes coded for pesticidal proteins were inserted. These crystal proteins originate from the bacterium Bacillus thuringiensis which lives in soil and they are toxic to several common maize pests like the corn rootworm and the European corn borer. Since its introduction, use of this type of transgenic maize has risen steadily until it now accounts for about 67% of the US acreage.

One potential environmental problem with these crops surrounds the unintentional release of the active proteins into the surrounding soil. They can escape via the roots or pollen, as well as from residual plant material left in the field after harvesting. It is important to be able to monitor the extent of these losses, because the proteins are toxic to nematodes and could also find their way into streams and rivers to threaten other organisms.

There have been a number of research investigations into the measurement of crystal proteins in soil but a team of US researchers from Southern Illinois University, Carbondale, noted that few of these had been validated. Moreover, the extraction process has not optimised. So, Michael Lydy, Sara Mueting and Katherine Strain undertook a full validation of the extraction and quantitation of the crystal protein Cry1Ab from soil.

Extraction assessment

Much of Illinois is given over to agriculture and 55% of the maize grown there is transgenic, containing at least one B. thuringiensis protein. So, the researchers collected three types of soil to test. One was topsoil collected during winter from a maize field in which no maize was present. The second originated from a field with no history of maize production and the third was a stream sediment from a forested region. They were chosen for their different properties, principally the proportions of clay, silt and sand and the ion exchange capacity.

The extraction buffer consisted of a solution of phosphate-buffered saline containing Tween and low concentrations of sodium and potassium chloride, sodium and potassium phosphate, and Tween 20 adjusted to pH 7.4 and it was used to extract Cry1Ab from fortified soils.

The extracts were analysed by ELISA using a commercial kit designed for B. thuringiensis Cry1Ab/1Ac proteins and the results were confirmed using dot blot assays with an anti-Cry1Ab/1Ac polyclonal antibody. The buffer effects and matrix effects were checked and validated before the actual levels of Cry1Ab in the soils were measured.

Matrix effects managed

The selected buffer caused no interferences with the ELISA test, as illustrated by successive dilution effects. However, one of the soils produced matrix interferences with protein signals appearing from a blank soil sample. The team attributed this effect to the high clay content.

This is consistent with published studies which have shown that the level of extraction decreases with an increasing content of organic matter. That was also the case here, with extraction efficiencies of 41, 74 and 89% correlating inversely with the amount of organic matter in the soil. Even though the recoveries were not the best, all of the soils gave consistent results with low coefficients of variation.

Following extensive buffer and matrix validation using matrix testing, dilution studies and fortification recoveries, the method was fully validated for the extraction and measurement of Cry1Ab in soils.

The pesticidal protein was detected at concentrations below 6.6 ng/g for some of the samples. Most of the positive soils were collected in July, so the protein release from maize could be related to the pollination process. No positive samples were found for samples taken in August and November, possibly due to protein degradation or increased binding to the clay component.

These levels were well below toxicological concentrations, implying that there are no risks to species that might be attacked by the protein. However, no toxicological studies or bioassays were carried out because of the reported lack of activity of the protein at these levels.

"The validation plan outlined in the present study utilized simple laboratory methodologies, and future studies should strongly consider using these validation methods to quantify transgenic proteins before publication,” the team concluded. “With controversy surrounding most research articles pertaining to genetically modified organisms, validated methods will ensure the consistency, precision, and accuracy of future studies."